Nucleic Acids Research Advance Access originally published online on January 21, 2009
Nucleic Acids Research 2009 37(6):1755-1766; doi:10.1093/nar/gkn1045
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2009, Vol. 37, No. 6 1755-1766
© Published by Oxford University Press (2009)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Molecular Biology |
Fidelity of plus-strand priming requires the nucleic acid chaperone activity of HIV-1 nucleocapsid protein
1Laboratory of Molecular Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, 2Department of Chemistry and Department of Biochemistry, The Ohio State University, Columbus, OH 43210, 3AIDS and Cancer Virus Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD 21702, USA and 4Shanghai Allist Pharmaceuticals, Zhangjiang, Shanghai 201203, People's Republic of China
* To whom correspondence should be addressed. Tel: +1 301 496 1970; Fax: +1 301 496 0243; Email: levinju{at}mail.nih.gov
Received September 18, 2008. Revised December 11, 2008. Accepted December 15, 2008.
During minus-strand DNA synthesis, RNase H degrades viral RNA sequences, generating potential plus-strand DNA primers. However, selection of the 3' polypurine tract (PPT) as the exclusive primer is required for formation of viral DNA with the correct 5'-end and for subsequent integration. Here we show a new function for the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NC) in reverse transcription: blocking mispriming by non-PPT RNAs. Three representative 20-nt RNAs from the PPT region were tested for primer extension. Each primer had activity in the absence of NC, but less than the PPT. NC reduced priming by these RNAs to essentially base-line level, whereas PPT priming was unaffected. RNase H cleavage and zinc coordination by NC were required for maximal inhibition of mispriming. Biophysical properties, including thermal stability, helical structure and reverse transcriptase (RT) binding affinity, showed significant differences between PPT and non-PPT duplexes and the trends were generally correlated with the biochemical data. Binding studies in reactions with both NC and RT ruled out a competition binding model to explain NC's observed effects on mispriming efficiency. Taken together, these results demonstrate that NC chaperone activity has a major role in ensuring the fidelity of plus-strand priming.